Inhibitors of plasmepsins

ABSTRACT

Compounds and methods for the inhibition of anti-malarial target aspartyl protease plasmepsins (e.g. Plasmepsin I, Plasmepsin II, Plasmepsin IV and HAP) are provided. The compounds are based on allophenylnorstatine substituted at positions R1-R4, such that R1 is isoquinoline, carboxyl, naphtalene, phenyl, phenol, benzene, an amino acid, and derivatives thereof; R2 and R3 are aliphatic groups; and R4 is indan, naphthalene, benzylamine, phenyl, phenol, cyclopentane, tert-butylamine, or derivatives thereof. The compounds may be used to inhibit Plasmepsin II, to kill malarial parasites, and to treat malaria in a patient. Certain of the substituted allophenylnorstatine-based compounds also exhibit inhibitory activity against Cathepsin D.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention generally relates to plasmepsin inhibitors and thetreatment of malaria. In particular, the invention provides methods andcompositions for the use of peptidomimetic allophenylnorstatine basedinhibitors of the antimalarial target aspartyl protease Plasmepsin II.More generally, the invention provides compounds and methods forinhibiting plasmepsins (e.g. Plasmepsin I, Plasmepsin II, Plasmepsin IV,HAP) which may provide a number of different pharmaceutical and medicalbenefits.

2. Background of the Invention

Malaria is one of the most serious infectious diseases in the world,affecting close to 300 million individuals each year. It has beenestimated that approximately 40% of the world population lives inregions where malaria is endemic. Each year between 1 and 1.5 millionpeople, mainly children, die from malaria, a number that is continuouslyincreasing due to the proliferation of parasites that are resistant toconventional drug therapies (Wyler, 1993). The rapid spread of drugresistant parasites clearly underscores the need for new therapies andconsequently the identification of novel targets for drug development.The malaria parasite uses the hemoglobin of the infected victim as asource of nutrients and energy. One of the key enzymes involved in thedegradation of hemoglobin is Plasmepsin II, an aspartic protease of 37kDa. Since the inhibition of this enzyme leads to starvation of theparasite, Plasmepsin II has been acknowledged to be an important targetfor the development of new antimalarials.

Four species of protozoan parasites of the genus Plasmodium (P.falciparum, P. vivax, P. malariae and P. ovale) are responsible formalaria in humans; P. vivax is the most common but P. falciparum causesthe most fatalities (Butler et al., 1997; Miller et al., 1994). ThePlasmodium parasite invades red blood cells and consumes tip to 75% oftheir hemoglobin content (Goldberg, 1993). The process takes place in anacidic digestive vacuole in the parasite. Three enzymes that digesthemoglobin have been identified in the food vacuole, one cysteineprotease (falcipain) and two aspartic proteases (Plasmepsin I andPlasmepsin II) (Francis et al., 1997b). The inhibition of any of theseenzymes leads to the starvation of the parasite and has been proposed asa viable strategy for drug development Plasmepsin I and Plasmepsin IIare 73% sequence identical. They have different substrate specificitiesand both contribute to the degradation of hemoglobin. Plasmepsin I issynthesized and processed to a mature form soon after the parasiteinvades the red blood cell, while the appearance of Plasmepsin II occurslater in development (Francis et al., 1997a). The expression andproduction of active recombinant Plasmepsin I has been shown to bedifficult, yielding a truncated protein that lacks the kineticproperties of the native enzyme (Luker et al., 1996). Plasmepsin U, onthe other hand, has been successfully expressed, the recombinant proteinbehaves identically to the protein isolated from the parasite and itshigh resolution structure has been determined by x-ray crystallography(Luker et al., 1996; Silva et al., 1996). For those reasons, PlasmepsinII is the target of choice for structure-based drug D5 design, eventhough the targeting of Plasmepsin I and other plasmepsins is alsoexpected.

Plasmepsin II is a protein of 37 kDa (329 amino acids). Thecrystallographic structure of Plasmepsin II in complex with the genericstatine-based aspartic protease inhibitor pepstatin A(IvaValValStaAlaSta) has been obtained at 2.7 Å for the Plasmodiumfalciparum enzyme (pdb file 1 sme) (Silva et al., 1996) and 2.5 Å forthe Plasmodium vivax enzyme (pdb file 1 qs8). Plasmepsin II has thetypical bilobal structure and topology of eukaryotic aspartic proteases.The active site is located at the interface between the two lobes and ispartially covered by a characteristic β-hairpin structure known as theflap. The secondary structure of Plasmepsin II is predominantly betawith only a small fraction (˜10%) of amino acids in alpha-helix. Eventhough pepstatin A and other related statine-containing peptides areknown to inhibit Plasmepsin II and other aspartic proteases, very fewnon-peptidic inhibitors have been described. A common problem with theseinhibitors is their poor selectivity and discrimination versus the humanaspartic protease Cathepsin D. Cathepsin D is a human protease in theendosomal-lysosomal pathway involved in lysosomal biogenesis and proteintargeting, it has 35% overall sequence homology and even higher bindingsite homology with Plasmepsin II, thus representing a target that needsto be avoided in the development of Plasmepsin II inhibitors.

Allophenylnorstatine-based compounds have been described before inrelation to the development of HIV-1 protease inhibitors (Kiso, 1996;Kiso, 1998; Kiso et al., 1999; Mimoto et al., 1999). These compounds arecharacterized by containing a unique unnatural amino acid,allophenylnorstatine ((2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid)containing a hydroxymethylcarbonyl isostere. Some of these compoundshave been shown to be high affinity inhibitors of the HIV-1 protease,they have low toxicity and excellent bioavailability (Kiso, 1996; Kiso,1998; Kiso et al., 1999; Mimoto et al., 1999).

SUMMARY OF THE INVENTION

The present invention provides compounds and methods for their use totreat malaria. The compounds work by inhibiting plasmepsins, aspartylproteases of the malarial parasite that are essential for the parasite'ssurvival. Examples of the Plasmepsins are, for example.

Plasmepsin 1, Plasmepsin II, Plasmepsin IV, and HAP. Methods are alsoprovided for inhibiting Plasmepsin proteases, and for killing malarialparasites.

The compounds themselves are synthesized based on theallophenylnorstatine “scaffolding” with various functional groups beingsubstituted at positions R1, R2, R3 and R4 of the structure. R1 is A orA-B, wherein A may be a linear or branched aliphatic hydrocarbon having1-7 carbon atoms which may be substituted by at least one carboxylgroup; a 6-membered monocyclic hydrocarbon which may be substituted witha substituent selected from the group consisting of alkyl amino,alkylamino, arylamino, hydroxy, alkyloxy and halogen atom; a bicyclichydrocarbon having 7-10 carbon atoms which may be substituted by asubstituent selected from the group consisting of alkyl, amino,alkylamino, arylamino, hydroxy, alkyloxy and halogen atom; or amonocyclic or bicyclic hydrocarbon wherein more than one carbon atom issubstituted; and B is —CO—NH—CH(Ra)—, —CH₂—CO—NH(Ra)—,—O—CH₂—CO—NH—CH(Ra)—, —OCH₂— and —CH₂O, where Ra is a linear or branchedaliphatic hydrocarbon having 1-7 carbon atoms that may be substitutedwith a substituent such as alkylthio, hydroxy, aromatic hydrocarbons,and carbamoyl; R2 is hydrogen or a linear or branched aliphatichydrocarbon having 1-6 carbons; R3 is hydrogen or a linear or branchedaliphatic hydrocarbon having 1-6 carbons; and R4 is a linear or branchedaliphatic hydrocarbon having 1-10 carbons which can be substituted witha substituent such as aryl, hydroxyl, alkyloxy, amino, alkylamino andhalogen; a monovalent moiety derived from an aromatic mono- or bicyclichydrocarbon having 12 or fewer carbons, and wherein said moiety can besubstituted by a substituent such as alkyl, aryl, hydroxyl, allkyloxy,amino, alkylamino, or halogen; a monovalent moiety derived from aheterocycle in which more than one carbon atom is substituted with ahetero atom, in which the moiety can be substituted by a substituentsuch as alkyl, aryl, hydroxyl, alkyloxy, amino, alkylamino, and halogen.

In preferred embodiments, A is: HOOC—CRbRb-CRbRb- wherein Rb is hydrogenor methyl; phenyl; 3-hydroxy-2-methylphenyl; 2,6-dimethylphenyl;3-chlorophenyl; 3-phenylaminophenyl; 3-dimethylaminophenyl; 1-naphtyl;2-naphtyl; 2-pyridyl; 5-isoquinolyl; 2-quinolyl; 2-benzofuranyl; and2-chromonyl; and B is —CO—NH—CH(Ra)—, —CH₂—CO—NH(Ra)—,—O—CH₂—CO—NH—CH(Ra)—, —OCH₂— or —CH₂O, wherein Ra is propyl, isopropyl,isobutyl, sec-butyl, methylthiomethyl, methylthioethyl,methylthiomethyl, phenylmethyl, carbamoylethyl, or 1-hydroxyethyl; R2 ishydrogen or methyl; R3 is hydrogen or methyl; and R4 is benzyl;tert-butyl; 2-hydroxybenzyl; 3-hydroxybenzyl; 4-hydroxybenzyl;2-hydroxyindanylyl; 2-hydroxy-1-phenethyl; 1-indanyl; 2-methoxybenzyl;3-methoxybenzyl; 4-methoxybenzyl; 4-methoxyphenethyl; 2-methylbenzyl;3-methylbenzyl; 4-methylbenzyl; naphtyl; and 1-phenethyl.

In yet another preferred embodiment, the allophenylnorstatine-basedcompound is a di-peptide and R1 is methylphenol, methylated derivativesof carboxyl, or chlorobenzene. Alternatively, theallophenylnorstatine-based compound may be a tri-peptide, in which caseR1 may be methylphenol, methylated derivatives of carboxyl,chlorobenzene, valine, leucine, isoleucine, methionine, phenylalanine,glutamine, or derivatives thereof. In preferred embodiments, R2 and R3are hydrogen, methyl or ethyl, and R4 is aminoindanol. In yet anotherembodiment, the allophenylnorstatine-based compound is a tri-peptideand: R1 is isoquinolineoxyacetyl at position P3 and methylthioalanine atposition P2, R2 and R3 are methyl, and R4 is (1S,2R)-aminoindanol ortert-butylamine.

In a preferred embodiment, the allophenylnorstatine-based compoundexhibits a Ki for Plasmepsin II from P. falciparum in the nanomolar tosubnanomolar range. Representative allophenylnorstatine-based compoundsare KNI-727, KNI-764, KNI-840, KNI-227, KNI-10006, KNI-10026, KNI-10033,KNI-10043 and KNI-10053. The plasmepsin which is inhibited may bePlasmepsin II that originates from a genus of Plasmodium, for example,P. falciparum, P. vivax, P. malariae or P. ovale.

The present invention also provides a method of killing malarialparasites by exposing them to an allophenylnorstatine-based compound ina quantity sufficient to inhibit at least one plasmepsin of the malarialparasite. The malarial parasite may be within a red blood cell and/orwithin a host, and may be Plasmepsin I, Plasmepsin II, Plasmepsin IV orHAP. The allophenylnorstatine-based compound may be substituted atpositions R1, R2, R3 and R4 as described above for inhibitingplasmepsins. Examples of the allophenylnorstatine-based compoundsinclude KNI-727, KNI-764, KNI-840, KNI-227, KNI-10006, KNI-10026,KNI-10033, KNI-10043 and KNI-10053. The malarial parasite may be from P.falciparum, P. vivax, P. malariae or P. ovale.

The present invention also provides a method of treating malaria whichcomprises administering a quantity of an allophenylnorstatine-basedcompound sufficient to alleviate the symptoms of malaria. Theallophenylnorstatine-based compound may be substituted at positions R1,R2, R3, and R4 as described above for inhibiting plasmepsins. Examplesof such compounds include KNI-727, KNI-764, KNI-840, KNI-227, KNI-10006,KNI-10026, KNI-10033, KNI-10043 and KNI-10053. In a preferredembodiment, the allophenylnorstatine-based compound has high selectivityfor plasmepsin rather than cathepsin D.

The present invention also provides a method of inhibiting the enzymecathepsin D, comprising, exposing the enzyme to anallophenylnorstatine-based compound. The allophenylnorstatine-basedcompound may be substituted at positions R1, R2, R3 and R4. R1 may be2,6-dimethylphenyl-OCH₂— or 5-isoquinolyl-O—CH₂—CO—NH—CH(Ra)— in whichRa is methylthiomethyl; R2 may be methyl or hydrogen; R3 may be methylor hydrogen: and R4 may be (1S, 2R)-2-hydroxyindanyl;(S)-2-hydroxy-1-phenethyl; (S)-indanyl; or (R)-1-phenethyl. Exemplarycompounds include KNI-391, KNI-10033, KNI-10006 and KNI-840.

The present invention also provides compositions of matter in the formof compounds KNI-10006 (FIG. 3J); KNI-10007 (FIG. 4A); KNI-10026 (FIG.4G); KNI-10031 (FIG. 5B); KNI-10033 (FIG. 5D); and KNI-10061, andKNI-10062 (FIGS. 10A and B).

BRIEF DESCRIPTION OF THE DRAWINGS AT 34 AT

FIG. 1. Chemical structures of: A, Phe-Leu, the cleavage motif ofplasmepsin II, and B, the allophenylnorstatine scaffold indicating theplaces (R1-R4) where different chemical functional groups can beintroduced.

FIG. 2. Chemical structures of: A, KNI-272; B, KNI-727; C, KNI-577; D,KNI-391; E, KNI-413; F, KNI-547; G, KNI-549; H, KNI-576; I, KNI-764; andJ, KNI-357.

FIG. 3. Chemical structures of: A, KNI-529; B, KNI-840; C, KNI-492; D,KNI-227; E, KNI-10001; F, KNI-10002; G, KNI-10003; H, KNI-10004; I,KNI-10005; and J, KNI-10006.

FIG. 4. Chemical structures of: A, KNI-10007; B, KNI-10008; C,KNI-10009; D, KNI-10010; E, KNI-10024; F, KNI-10025; G, KNI-10026; H,KNI-10027; I, KNI-10028; and J, KNI-10029.

FIG. 5. Chemical structures of: A, KNI-10030; B, KNI-10031; C,KNI-10032; D, KNI-10033; E, KNI-10041; F, KNI-10042; G, KNI-10043; H,KNI-10044; I, KNI-10045; and J, KNI-10046.

FIG. 6. Chemical structures of: A, KNI-10047; B, KNI-10048; C,KNI-10049; D, KNI-10050; E, KNI-10051; F, KNI-10052; G, KNI-10053; Hl,KNI-10054; I, KNI-10055; J, KNI-10056; and K, KNI-10057.

FIG. 7A-C. Inhibition of Plasmepsin II from Plasmodium falciparum byKNI-10033, (NI-10030 and KNI-10006 at 20° C. in 10 mM sodium citrate,100 mM NaCl, 2% DMSO, pH 4.0. The inhibition constants are 3 nM, 11 nMand 0.5 rLM respectively.

FIGS. 8A and B. A and B: KNI inhibitors with high affinity forPlasmepsin II and high selectivity between Plasmepsin II and humanCathepsin D. A, KNI-227 and B, KNI-727 have K_(i)s of 30 and 70 nMagainst Plasmepsin II and Cathepsin D selectivities of 50 and 22respectively.

FIG. 9. Percent of malarial parasites killed in malaria infected humanerythrocytes by KNI-727, KNI-764 and KNI-840. Analysis of the datayields an IC50 of 5.7 mM for KNI-764 and 10 mM for KNI-727 and KNI-840respectively.

FIGS. 10A and B. Structural representations of A, KNI-10061 and B,10062.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The present invention is based on Applicant's discovery thatallophenylnorstatine-based compounds are powerful and selectiveinhibitors of Plasmepsin aspartyl proteases, which are superbanti-malarial targets. The allophenylnorstatine core (FIG. 1B) resemblesthe Phe-Leu motif (FIG. 1A) that defines the main substrate forplasmepsin II in the human hemoglobin molecule.

The present invention thus provides a method of inhibiting plasmepsins(e.g. the enzyme plasmepsin II) by exposing the enzyme to at least oneallophenylnorstatine-based compound. By allophenynorstatine-basedcompound we mean a compound with the basic “scaffoldinig” structuredepicted in FIG. 1B. Compounds which are based on this structure arethose which have been synthesized with various moieties or functionalgroups substituted at positions R1-R4 of the scaffolding. Depending onthe exact nature of the substitutions, the resulting compound may be di-or tri-peptidic in nature. Using standard enzymatic nomenclature, if theinhibitor is tripeptidic in nature, then the allophenylnorstatine moietyin these compounds corresponds to the P1 position, R1 corresponds to theP2 position, the thioproline group together with R12 and R3 correspondto the P1′ position, and R4 corresponds to the P2′ position. If theinhibitor is tripeptidic, either by virtue of R1 being substituted withan amino acid or with a group that, in bonding to the R1 site, forms athird peptide bond, then R1 may correspond to both the P2 and P3positions.

Applicants have discovered that, in particular, the followingsubstitutions yield allophenylnorstatine compounds with especiallyuseful plasmepsin inhibitory characteristics:R1 is A or A-B, wherein Amay be a linear or branched aliphatic hydrocarbon having 1-7 carbonatoms which may be substituted by at least one carboxyl group; a6-membered monocyclic hydrocarbon which may be substituted with asubstituent selected from the group consisting of alkyl, amino,alkylamino, arylamino, hydroxy, alkyloxy and halogen atom; a bicyclichydrocarbon having 7-10 carbon atoms which may be substituted by asubstituent selected from the group consisting of alkyl amino,allcylamino, arylamino, hydroxy, alkyloxy and halogen atom; or amonocyclic or bicyclic hydrocarbon wherein more than one carbon atom issubstituted; and B is —CO—NH—CH(Ra)—, —CH₂—CO—NH(Ra)—,—O—CH₂CO—NH—CH(Ra)—, —OCH₂— and —CH₂O, where Ra is a linear or branchedaliphatic hydrocarbon having 1-7 carbon atoms that may be substitutedwith a substituent such as alkylthio, hydroxy, aromatic hydrocarbons,and carbamoyl; R2 is hydrogen or a linear or branched aliphatichydrocarbon having 1-6 carbons; R3 is hydrogen or a linear or branchedaliphatic hydrocarbon having 1-6 carbons; and R4 is a linear or branchedaliphatic hydrocarbon having 1-10 carbons which can be substituted witha substituent such as aryl, hydroxyl, alkyloxy, amino, alkylamino andhalogen; a monovalent moiety derived from an aromatic mono- or bicyclichydrocarbon having 12 or fewer carbons, and wherein said moiety can besubstituted by a substituent such as alkyl, aryl, hydroxyl, alkyloxy,amino, alkylamino, or halogen; a monovalent moiety derived from aheterocycle in which more than one carbon atom is substituted with ahetero atom, in which the moiety can be substituted by a substituentsuch as alkyl, aryl, hydroxyl, alkyloxy, amino, alkylamino, and halogen.

In preferred embodiments, A is: HOOC—CRbRb-CRbRb- wherein Rb is hydrogenor methyl; phenyl; 3-hydroxy-2-methylphenyl; 2,6-dimethylphenyl;3-chlorophenyl; 3-phenylaminophenyl; 3-dimethylaminophenyl; 1-naphtyl;2-naphtyl; 2-pyridyl; 5-isoquinolyl; 2-quinolyl; 2-benzofuranyl; and2-chromonyl; and B is —CO—NH—CH(Ra)—, —CH₂—CO—NH(Ra)—,—O—CH₂—CO—NH—CH(Ra)—, —OCH₂— or —CH₂O, wherein Ra is propyl, isopropyl,isobutyl, sec-butyl, methylthiomethyl, methylthioethyl, ethylthiomethyl,phenylmethyl, carbamoylethyl, or 1-hydroxyethyl; Rat is hydrogen ormethyl; R3 is hydrogen or methyl; and R4 is benzyl; tert-butyl;2-hydroxybenzyl; 3-hydroxybenzyl; 4-hydroxybenzyl; 2-hydroxyindanylyl;2-hydroxy-1-phenethyl; 1-indanyl; 2-methoxybenzyl; 3-methoxybenzyl;4-methoxybenzyl; 4-methoxyphenethyl; 2-methylbenzyl; 3-methylbenzyl;4-methylbenzyl; naphtyl; and 1-phenethyl.

Those of skill in the art will recognize that, by the term “derivatives”we mean functional groups which are modified or substituted by theaddition of one or more of certain other typically low (e.g. about MW100 or less) molecular weight species e.g. by the addition of methyl,benzyl, amine, hydroxyl, acetyl, halogen, thiol, alkyl, aryl, alkyloxy,alkylamino, carbamoyl, carboxyl groups and the like. Similarly, acompound that is “derived from” is one that is the result of such amodification. Specific examples of moieties or functional groups whichmay be substituted at position R1 include but are not limited to:methylphenol, methylated variations of a carboxylic acid group,isoquinoline, naphthalene, chlorobenzene and phenyl in both di- andtri-peptide mimetics; and valine, isoleucine, methionine, leucine,phenylalanine, and glutamine in tri-peptide mimetics. Examples ofmoieties or functional groups which may be substituted at position R2include but are not limited to: hydrogen, methyl and ethyl, moieties.Functional groups which may be substituted at position R3 include butare not limited to: hydrogen, methyl and ethyl. Moieties or functionalgroups which may be substituted at position R4 include but are notlimited to: various benzylamine derivatives, aminoindanol,tert-butylamine, phenol, naphthalene, cyclopentane and indan.

In a preferred embodiment of the present invention, the groups which aresubstituted at position R1 are as follows: a P3 moiety ofisoquinolineoxyacetyl and a P2 moiety of methylthioalanine in the caseof tri-peptide mimetics, or a P2 moiety of dimethylphenoxyacetyl ormethylphenol in the case of di-peptide mimetics. In a preferredembodiment of the present invention, the group which is substituted atpositions R2 and R3 is a methyl group. In a preferred embodiment of thepresent invention, the groups which are substituted at position R4 are(1S, 2R)-aminoindanol or tert-butylamine.

Those of skill in the art will recognize that the methods forsynthesizing such compounds are well-established and accessible. Forexample, see Kiso, 1996; Mimoto et al., 1999.

Specific examples of the compounds which are substituted in this mannerinclude those which exhibit a particularly low Ki value (e.g. KNI-10006,KNI-10030, KNI-10033) and those which exhibit a particularly low Kivalue and high selectivity or discrimination for Plasmepsin II comparedto Cathepsin D (e.g. KNI-727, KNI-227).

In a preferred embodiment of the present invention, the standard“Plasmepsin II” for determining Ki values is derived from Plasmodiumfalciparum. i.e. the protease is the product of a gene which wasoriginally cloned from Plasmodium falciparum. However, those of skill inthe art will recognize that the exact source of the Plasmepsin II whichis utilized to establish Ki values of the compounds of the presentinvention is not a key feature of the invention in that Plasmepsin IIfrom a different Plasmodium species, or from various strains of thespecies, could also be utilized. Any art-recognized Plasmepsin II may beutilized to establish Ki values, so long as a useful correlation may bemade between the Ki values obtained and the efficacy of the compound inachieving desirable ends such as killing malaria parasites and treatingpatients to alleviate symptoms of malaria.

By “low Ki value” we mean a Ki value for the inhibition of Plasmepsin IIin the range of picomolar to nanomolar. In a preferred embodiment of thepresent invention, the Ki value for the compound will be, for exampleless than about 100 nmolar, and more preferably, less than about 50nmolar, and even more preferably less than about 10 nmolar.

By having high selectivity or a high “discrimination factor” forPlasmepsin II vs Cathepsin D, we mean that the Ki of the compound forCathepsin D is at least about 2 fold higher than the Ki of the compoundfor Plasmepsin II. In a preferred embodiment, the ratio of the Ki's ofCathepsin D to Plasmepsin II is about at least 4, and more preferablyabout at least 10 or more.

However, those of skill in the art will recognize that the desirabilityof a compound exhibiting high selectivity for Plasmepsin II can beoffset or compensated for if the compound displays a low Ki forPlasmepsin II. This is because the lower the Ki of a compound forPlasmepsin II, the less of the compound must be administered and/or overa shorter period of time, in order to achieve a desired effect, such asinhibiting the enzyme or killing a malarial parasite. Logically, if avery low dose of an inhibitor can be administered, then the side effects(e.g. the inhibition of Cathepsin D) will also be decreased, regardlessof the level of selectivity of the compound for Plasmepsin II. Forexample, compound KNI-10006 displays only a 4-fold “discriminationfactor”, i.e. the compound has about a 4-fold higher affinity forPlasmepsin II than for Cathepsin D. However, because the Ki is very low(0.5 nmolar), very little of the compound will need to be administeredin order to achieve a desired result. Thus, side effects such as theinhibition of Cathepsin D will be minimized. In contrast, KNI-840 alsohas a discrimination factor of 4, but has a Ki of about 20, which couldnecessitate the administration of about 40 times as much compound(0.5×40=20) to achieve the same level of inhibition as KNI-10006.

In general, for compounds of the present invention which exhibit Ki'sfor Plasmepsin II in the range of less than about 10 nanomolar, adiscrimination factor may not play an essential role in the efficacy ofthe compound. For compounds with a Ki for Plasmepsin II in the range ofabout 10 nmolar to about 100 nmolar, a discrimination factor in therange of about 10 to about 50 should be acceptable.

In a preferred embodiment, the present invention provides methods ofinhibiting the enzyme Plasmepsin II. However, the commentary hereinconcerning Plasmepsin H is similar for other plasmepsins, such as thosedescribed in Coombs, et al., 2002. By “Plasmepsin II” we mean theaspartyl protease Plasmepsin 11 originating from any protozoan parasiteof the genus Plasmodium, for example, from P. falciparum, P. vivax, P.malariae or P. ovale. By “originating from” we mean that the enzyme was,for example, first detected in, isolated from, or cloned from thatorganism, regardless of later genetic manipulations. In a preferredembodiment of the present invention, the Plasmepsin II that is inhibitedis that of P. falciparum. Further, the enzyme to be inhibited may beeither naturally occurring within the parasite within a host, naturallyoccurring within the parasite in a laboratory culturing system, or mayhave been obtained from the parasite by isolation techniques which arewell known to those of skill in the art, or may be obtained from arecombinant gene which encodes the protein. In addition, the term“Plasmepsin II” is meant to include all forms of the protease such asthose displaying mutations in the gene (which may or may not bereflected in the primary structure of the protein, such as innaturally-occurring nucleotide polymorphisms) as a result of naturallyoccurring mutations, variation between and within species or strains,including other genomically-identified plasmepsins, or purposeful (i.e.intentional) or fortuitous mutations which are introduced in alaboratory setting, e.g. during recombinant cloning of the gene. As suchforms of Plasmepsin II may be inhibited by the practice of the presentinvention. In general, any aspartyl protease displaying about 75% orgreater, or preferably from about 80% or greater, or most preferablyfrom about 90% or greater amino acid sequence homology with PlasmepsinII from P. falciparum, may be inhibited by the methods of the presentinvention, so long as the substrate specificity of such a protease isclose enough to that of Plasmepsin II of P. falciparum so that thecompounds of the present invention inhibit the protease.

In a preferred embodiment, the allophenylnorstatine-based compound whichis utilized to inhibit the Plasmepsin II is substituted at positionsR1-R4 such that R1 is 2,6-dimethylphenyl-OCH₂—, 3-hydroxy-2-methylphenylor 5-isoquinolyl-O—CH₂—CO—NH—CH(Ra) where Ra is methylthiomethyl; R2 ismethyl; R3 is methyl; and R4 is benzyl; tert-butyl; 2-hydroxybenzyl;3-hydroxybenzyl; (1S, 2R)-2-hydroxyindanyl; (1R,2S)-2-hydroxyindanyl;(S)-2-hydroxy-1-phenethyl; (S)-indanyl; 4-methoxyphenethyl;2-methylbenzyl; 3-methylbenzyl; naphthyl; or (R)-1-phenethyl.

In yet another preferred embodiment, the allophenylnorstatine-basedcompound winch is utilized to inhibit the Plasmepsin II is substitutedat positions R1-R4 such that R1 is isoquinolineoxyacetyl andmethylthioalanine in tri-peptides and dimethylphenoxyacetyl ormethylphenol in di-peptides, R2 is a methyl group, R3 is a methyl group,and R4 is (1S, 2R)—aminoindanol or tert-butylamine, indan, methylphenyl,or o-benzylamine and the allophenylnorstatine-based compound exhibits aKi for Plasmepsin II from P. falciparum in the nanomolar to subnanomolarrange. In yet another preferred embodiment, theallophenylnorstatine-based compound is KNI-727, KNI-764, KNI-840,KNI-227, KNI-10006, KNI-10026, KNI-10033, KNI-10043 or KNI-10053, andthe Plasmepsin II originates from a genus of Plasmodium selected fromthe group consisting of P. falciparum, P. vivax, P. malariae or P.ovale.

The present invention provides methods for treating malaria in a patientin need thereof in order to alleviate symptoms of the disease byadministering an allophenylnorstatine-based compound to the patient. Ina preferred embodiment, the allophenylnorstatine-based compound which isutilized to treat malaria is substituted at positions R1-R4 as describedabove for inhibiting plasmepsins. In yet another embodiment, R1 isisoquinolineoxyacetyl and methylthioalanine in tri-peptides anddimethylphenoxyacetyl or methylphenol in di-peptides, R2 is a methylgroup, R3 is a methyl group, and R4 is (1S, 2R)-aminoindanol ortert-butylamine, indan, methylphenyl, or o-benzylamine, and theallophenylnorstatine-based compound exhibits a Ki for Plasmepsin II fromP. falciparum in the nanomolar to subnanomolar range. In yet anotherpreferred embodiment, the allophenylnorstatine-based compound which isutilized is KNI-727, KNI-764, KNI-840, KNI-227, KNI-10006, KNI-10026,KNI-10033, KNI-10043 or KNI-10053. The in vivo IC50 for such a compoundwill generally be in the range of about 0.1 to about 100 μM andpreferably below about 10 μM.

Those of skill in the art will recognize that the precise quantity ofsuch a compound to be administered will vary from case to case, and isbest determined by a skilled practitioner such as a physician. Forexample, the amount may vary based on several characteristics of thepatient, e.g. age, gender, weight, overall physical condition, extent ofdisease, and the like. Further, the individual characteristics of thecompound itself (e.g. Ki, selectivity, IC50, solubility,bioavailability, and the like) will also play a role in the amount ofcompound that must be administered. However, in general, the requiredamount will be such that the concentration of compound in the bloodstream of the patient is about equal to the IC50 of the compound. Inpreferred embodiment of the present, this concentration will be in therange of about 0.1 to about 100 μM, and more preferably below about 10μM.

The causative agent of the malaria that is treated in a patientaccording to the methods of the present invention may be any of avariety of Plasmodium species which are well-known to those of skill inthe art, including but not limited to P. falciparum, P. vivax, P.malariae or P. ovale, as well as various strains of these species.Further, the species may or may not be already resistant to other formsof treatment, such as treatment with chloroquine.

The compounds which are administered in the practice of the presentinvention may be administered in any of many forms which are well-knownto those of skill in the art. For example, they may be administered inany of a variety of art-accepted forms such as tablets, capsules,various injectable formulations, liquids for oral administration and thelike, as suitable for the desired means of administration. Thepreparation which is administered may include one or more than oneinhibitory compound, and may further contain other suitable substancesand excipients, including but not limited to physiological acceptablebuffering agents, stabilizers (e.g. antioxidants), flavoring agents,agents to effect the solubilization of the compound, and the like.Administration of the compounds may be effected by any of a variety ofroutes that are well-known to those of skill in the art, including butnot limited to oral, perenteral, intravenously, via inhalation, and thelike. Further, the compounds may be administered in conjunction withother appropriate treatment modalities, for example, with nutritionalsupplements, agents to reduce symptoms such as fever, treatment withother anti-malarial agents such as chloroquine, mefloquine,sulphadoxine-pyrimethamine, fansidar, artemisinin, quinine,atovaquone-proguanil, and the like

The present invention also provides methods for killing malarialparasites. Malarial parasites which may be killed by the methods of thepresent invention include but are not limited to P. falciparum, P.vivax, P. malariae or P. ovale., and various strains thereof. The methodis carried out by exposing the parasite to an allophenylnorstatine-basedcompound in a quantity sufficient to kill the parasite. In preferredembodiments, the allophenylnorstatine-based compound is substituted atpositions R1-R4 such that R1 is isoquinolineoxyacetyl andmethylthioalanine in tri-peptides and dimethylphenoxyacetyl ormethylphenol in di-peptides, R2 is a methyl group, R3 is a methyl group,and R4 is (1S, 2R)-aminoindanol or tert-butylamine, indan, methylphenyl,or o-benzylamine. The parasite to be killed may be within a host, or maybe in culture. The parasite may be within a red blood cell. In apreferred embodiment, the allophenylnorstatine-based compound used tokill the malarial parasite exhibits a Ki for Plasmepsin II from P.falciparum in the nanomolar to subnanomolar range, and may be KNI-727,KNI-764, KNI-840, KNI-227, KNI-10006, KNI-10026, KNI-10033, KNI-10043 orKNI-10053. The IC50 for such a compound will generally be in the rangeof about 0.1 to 1100 M and preferably below about 10 μM.

The quantity of allophenylnorstatine-based compound required to kill amalarial parasite (and by extension, to treat malaria) can beascertained in part by determining the IC50 of the compound. Those ofskill in the art will recognize that, while in general it may beexpected that the IC50 of a compound will be related to its affinity forthe enzyme Plasmepsin II, it is also possible that compounds with lesserKi values will exhibit IC50 values that are more favorable thananticipated based on their Ki values. For example, see the discussiongiven in Example 5. Such results may be due to any of several factors,such as the ability of the compound to access the targeted protease(i.e. to enter red blood cells), differences in solubility and ADME(absorption, distribution, metabolism and excretion), or other factors.In general, for utilization in the practice of the present invention, anIC50 value in the range of about 0.1 to about 100 μM and preferablybelow about 10 μM.

The present invention also provides new compositions of matter. Thecompositions of matter are allophenylnorstatine-based compounds whichare useful for carrying out the methods of the present invention such asinhibiting the plasmepsins, killing malarial parasites, and treatingmalaria in a patient. The new compositions are represented in thisapplication as: KNI-10006 (FIG. 3J); KNI-10007 (FIG. 4A); KNI-10026(FIG. 4G); KNI-10031 (FIG. 5B); KNI-10033 (FIG. 5D); and KNI-10061, andKNI-10062 (FIG. 10A and B).

It is noteworthy that some of the allophenylnorstatine-based compoundsare also potent inhibitors of the human enzyme Cathepsin D. Thus, theycan be used in applications in which this enzyme needs to be inhibited.The R1-R4 substitution pattern for allophenylnorstatine-based compoundsthat are good inhibitors of Cathepsin D is: R1 may be2,6-dimethylphenyl-OCH₂— or 5-isoquinolyl-O—CH₂—CO—NH—CH(Ra)— in whichRa is methylthiomethyl; R2 may be methyl or hydrogen; R3 may be methylor hydrogen; and R4 may be (1S, 2R)-2-hydroxyindanyl;(S)-2-hydroxy-1-phenethyl; (S)-indanyl; or (R)-1-phenethyl. In apreferred embodiment, when the allophenylnorstatine-based compound is adipeptide R1 is carboxyl or a derivative of a carboxyl at position P2and when said allophenylnorstatine-based compound is a tripeptide R1 ischromen-4-one at position P3 or valine at position P3; both R2 and R3are hydrogen or methyl; and R4 is indanol or tert-butylamine. Specificexamples of these compounds are high specificity compounds such asKNI-391 and high affinity compounds such as KNI-10033, the Ki's forCathepsin D for which are 1.35 and 0.040 μM, respectively, as well asKNI-10006 and KNI-840. In general, in order to be useful as an inhibitorof Cathepsin D, a compound will have a Ki in the picomolar to thenanomolar range.

Applications in which the inhibition of Cathepsin D is useful includebut are not limited to the analysis and monitoring of proteincatabolism, antigen processing, degenerative diseases, and breast cancerprogression.

EXAMPLES Example 1

The specific activity of plasmepsin II from Plasmodium falciparum (PlmII) cloned in and purified from Escherchia coli and commerciallyavailable human Cathepsin D were measured by following the hydrolysis ofthe chromogenic substrateAla-Leu-Glu-Arg-Thr-nPhe-Phe-Ser-Phe-Pro-Thr-OH (California PeptideResearch Inc., Napa, Calif.). The decrease in absorbance upon hydrolysisis monitored at 300 nm. Typical Plasmepsin II preparations hydrolyzechromogenic substrate at 4-5 s⁻¹ at 37° C. The inhibition assays wereperformed at 25° C. in 10 mM sodium formate, 2% DMSO, pH 4.0. These pHconditions mimic the conditions in the food vacuole of the parasite.Under the conditions of the assays the K_(m) for plasmepsin II is 20 μMand for human Cathepsin D is 130 μM. Inhibition constants (K_(i)) forthe inhibitors are obtained at the desired temperature and solventconditions by measuring the rate of substrate hydrolysis at increasingamounts of inhibitors. Exemplary results from three compounds(KNI-10033, KNI-10030 and KNI-10006) are shown in FIG. 7A-C, and theresults obtained with all KNI compounds tested are given in Table 1.TABLE 1 Allophenylnorstatine-based Compounds Tested for Plasmepsin IIInhibition* K_(i) Plm II K_(i) CatD Compound MW μM μM 1 KNI-272 668 1.0± 0.2 2.58 ± 0.30 2 KNI-727 556 0.070 ± 0.040 1.60 ± 0.30 3 KNI-577 5280.590 ± 0.070 3.44 ± 0.70 4 KNI-391 494 12.66 ± 1.50  1.35 ± 0.08 5KNI-413 522 1.39 ± 0.50 7.26 ± 1.40 6 KNI-547 494 30.7 ± 8.1  18.17 ±3.30  7 KNI-549 522 8.60 ± 1.30 37.68 ± 8.20  8 KNI-576 500 9.26 ± 3.607.03 ± 1.60 9 KNI-764 576 0.030 ± 0.009 0.201 ± 0.020 10 KNI-357 46696.87 ± 19.1  8.12 ± 1.50 11 KNI-529 668 8.09 ± 2.60 8.82 ± 2.80 12KNI-840 604 0.020 ± 0.010 0.080 ± 0.090 13 KNI-492 678 0.34 ± 0.02 3.70± 0.35 14 KNI-227 696 0.036 ± 0.01  1.773 ± 0.23  15 KNI-10001 692 0.359± 0.060 1.609 ± 0.330 16 KNI-10002 665 0.244 ± 0.02  0.112 ± 0.020 17KNI-10003 598 1.148 ± 0.190 0.97 ± 0.10 18 KNI-10004 648 0.522 ± 0.0500.290 ± 0.090 19 KNI-10005 718 0.250 ± 0.190 0.507 ± 0.100 20 KNI-10006632 0.0005 ± 0.001  0.002 ± 0.010 21 KNI-10007 632 0.071 ± 0.010 0.806 ±0.600 22 KNI-10008 620 0.169 ± 0.020 0.461 ± 0.140 23 KNI-10009 6372.183 ± 0.250 0.515 ± 0.047 24 KNI-10010 670 0.691 ± 0.040 5.66 ± 0.6125 KNI-10024 692 0.131 ± 0.040 0.28 ± 0.05 26 KNI-10025 634 0.075 ±0.010 0.655 ± 0.083 27 KNI-10026 616 0.015 ± 0.005 0.105 ± 0.018 28KNI-10027 647 1.074 ± 0.090 1.034 ± 0.200 29 KNI-10028 661 1.628 ± 0.1570.69 ± 0.07 30 KNI-10029 661 0.860 ± 0.090 0.282 ± 0.039 31 KNI-10030604 0.011 ± 0.003 0.111 ± 0.015 32 KNI-10031 626 0.083 ± 0.007 0.130 ±0.019 33 KNI-10032 710 0.535 ± 0.055 2.62 ± 0.39 34 KNI-10033 772 0.003± 0.001 0.040 ± 0.010 35 KNI-10041 673 0.496 ± 0.069 1.668 ± 0.256 36KNI-10042 678 0.222 ± 0.017 2.263 ± 0.166 37 KNI-10043 620 0.022 ± 0.0050.053 ± 0.008 38 KNI-10044 620 0.532 ± 0.070 1.040 ± 0.178 39 KNI-10045562 1.084 ± 0.165 6.158 ± 0.671 40 KNI-10046 590 0.096 ± 0.020 0.260 ±0.023 41 KNI-10047 606 0.055 ± 0.007 0.252 ± 0.053 42 KNI-10048 6060.099 ± 0.014 0.771 ± 0.097 43 KNI-10049 627 1.511 ± 0.125 2.580 ± 0.68244 KNI-10050 680 2.625 ± 0.281 38.10 ± 1.87  45 KNI-10051 726 0.186 ±0.014 6.512 ± 1.224 46 KNI-10052 604 0.572 ± 0.054 1.321 ± 0.152 47KNI-10053 604 0.041 ± 0.007 0.360 ± 0.054 48 KNI-10054 606 0.273 ± 0.0411.642 ± 0.146 49 KNI-10055 707 1.172 ± 0.071 6.279 ± 0.981 50 KNI-10056627 1.773 ± 0.154 1.283 ± 0.161 51 KNI-10057 710 0.428 ± 0.021 5.295 ±0.633*Inhibition constants (K_(i)) were obtained by fitting the data to thestandard equations for competitive inhibition.

As can be seen, the inhibition results indicate that several of thecompounds, KNI-727, KNI-764, KNI-840, KNI-227, KNI-10006, KNI-10026,KNI-10033, KNI-10043 and KNI-10053 have inhibition constants againstPlasmepsin 11 in the nanomolar and subnanomolar range (also see Table 2,column 2). This Example demonstrates that allophenylnorstatine-basedcompounds that are substituted such that R1 is isoquinolineoxyacetyl andmethylthioalanine in tri-peptides and dimethylphenoxyacetyl ormethylphenol in di-peptides, R2 is a methyl group, R3 is a methyl group,and R4 is (1S, 2R)-aminoindanol or tert-butylamine, indan, methylphenyl,or o-benzylamine, are powerful inhibitors of Plasmepsin II.

Example 2 Ratio of Inhibition Constants for Plasmepsin II VersusCathepsin D

As described above, it is preferable that the inhibitors display notonly strong inhibition of Plasmepsin II, but that they displayselectivity for that enzyme. The results of a comparison of inhibitioncurves for two compounds, KNI-227 and KNI-727, are given in FIGS. 8A andB. Column 4 of Table 2 gives the numerical values of a comparison of theinhibition constants of the indicated KNI-compounds for Plasmepsin 11versus Cathepsin D. The values given represent the ratio of the Ki forCathepsin D to the for Plasmepsin II, i.e. the discrimination factor. Ascan be seen, the selected KNI compounds show a range of from about 2 toabout 50 fold selectivity for Plasmepsin II over Cathepsin D.

This example demonstrates that KNI compounds have the ability tostrongly inhibit plasmepsin II while discriminating against Cathepsin Din order to achieve high selectivity. TABLE 2 Comparison of K_(i)'s forPlasmepsin II and Cathepsin D Discrimination Factor: Fold K_(i) K_(i)Selectivity for Figure Plasmepsin II Cathepsin Plasmepsin II ReferenceCompound (nM) D (nM) over Cathepsin D Number KNI-727 70 1600 ˜23 KNI-76430 201 ˜7 KNI-840 20 80 4 KNI-227 36 1773 ˜49 KNI-10006 0.5 2 4KNI-10026 15 105 7 KNI-10030 11 111 ˜10 KNI-10033 3 40 ˜13 KNI-10043 2253 2.4 KNI-10053 41 360 ˜9

Example 3 Evaluation of the Ability of KNI Compounds to Kill the MalariaParasite

The ability of some KNI compounds to kill the malaria parasite wasevaluated by measuring the IC50 in a malaria-infected human red bloodcell assay. Activity was determined by measuring the incorporation of[³H]hypoxanthine. Briefly, chloroquine-sensitive Plasmodium falciparum(NF54) were maintained in a 2.4% suspension of type O+human erythrocytesin RPMI 1640, supplemented with 25 mM HEPES, 27 mM NaHCO₃, and 10%heat-inactivated human type O⁺ serum, under 3% O₂, 4% CO₂, and 93% N₂.20 mM stock solutions of KNI-764 and KNI-727 and KNI-840 were preparedin DMSODMSO solutions were diluted 500 fold in medium, serially dilutedin 0.2% DMSO in medium, then 100 μl aliquots were pipetted intomicrotiter plate wells. Provisional EC₅₀ values were obtained in asurvey of ten 5-fold dilutions yielding final concentrations (inquadruplicate) of 0.00001-20 μM. Plates included 8 wells of no drugcontrols (4 with and 4 without DMSO) and 4 wells in uninfectederythrocytes. Parasite culture (0.25% parasitemia in 2.3% hematocrit,100 μl per well) was added and the plate was incubated for 48 hoursprior to the addition of 0.6 μCi [³H]hypoxanthine and subsequent 20 hincubation. Cells were harvested onto GF-C glass filters. The filterswere washed four times with 3 ml water per sample spot, dried under aheat lamp, and counted in scintillation cocktail. Decays per minutevalues were downloaded and analyzed in order to yield the mean andstandard deviation at each drug concentration. Dose-response curves werefit to the experimental data in order to obtain the drug concentrationthat kills 50% of parasites (IC50). The results obtained for KNI-727,KINI-840 and KNI-764 are shown in FIG. 9.

As can be seen, KNI-727, KNI-764, and KNI-840 all are able to kill themalaria parasite with low IC50 values of 5.7 μM, 10 μM and 10 μM,respectively. This example demonstrates that KNI compounds are able tokill malaria parasites in infected human erythrocyte cultures.

Example 4 The Binding Energetics of KNI Compounds

The allophenylnorstatine scaffold was chosen as a prospective plasmepsininhibitor because of its close similarity to the primary cleavage siteof plasmepsin II, the peptide bond between Phe 33 and Leu 34 in thealpha chain of hemoglobin, and the potential for the introduction ofdifferent types of functional groups (FIG. 1B). The allophenylnorstatinescaffold contains four different positions (labeled R1-R4 in thestructure in FIG. 1B) where different chemical functional groups can beintroduced in order to improve binding affinity and selectivity. Usingthe standard enzymatic nomenclature, the allophenylnorstatine moiety inthese compounds corresponds to the P1 position, R1 corresponds to the P2position, the thioproline group together with R2 and R3 correspond tothe P1′ position and R4 corresponds to the P2′ position.

The binding energetics of a library of allophenylnorstatine-basedcompounds (KNI library) have been measured as described above. Thebinding enthalpy of a subset of these compounds was determined highsensitivity isothermal titration calorimetry. All the samples were foundto bind plasmepsin It with a favorable binding enthalpy ranging from−1.5 to −7.4 kcal/mol. The binding of KNI compounds to Plasmepsin II isboth enthalpically and entropically favorable. This is a very importantcharacteristic from the point of view of further optimization of thesecompounds since it reflects the fact that not a single type of force(enthalpy or entropy) is driving the binding reaction as is the casewith other inhibitors (Todd et al., 2000). Also, a well balanceddistribution of binding forces lowers the susceptibility of inhibitorsto changes in the environment or in the target molecule, thus makingthem less prone to drug resistance issues. By displaying a favorablebinding enthalpy, the binding entropy does not have to exhibit extremevalues in order to achieve high binding affinity. The implication isthat the compounds can be allowed to have certain flexibility thatallows them to accommodate to variations in the target such as thosefound in drug-resistant mutants or naturally-occurring amino acidpolymorphisms among the plasmepsins.

Example 5 Comparison with Previously Reported Compounds

The Ki of KNI-10006 for Plasmepsin II is 0.5 mM, being the highest sofar. The K_(i) of KNI-227 and KNI-727 for plasmepsin II are 30 and 60 nMwith a discrimination factor of 50 and 22 with regard to Cathepsin D. Inaddition, KNI-727 has an IC50 of 10 μM measured in the malaria-infectedred blood cell assay. Previously Haque et al. (Haque et al., 1999)reported results on a series of plasmepsin II inhibitors. The bestinhibitor had a K_(i) of 4 nM, however the discrimination factor betweenplasmepsin II and Cathepsin D was only 14. Silva et al (Silva et al.,1996) reported on a peptide-based inhibitor with a K_(i) of 0.6 nM and adiscrimination factor of 35, however the reported IC50 in a red bloodcell assay was around 20 μM. The discrimination factor for KNI-227 andKNI-727 are also significantly better than the discrimination factor of6.4 obtained with the best inhibitor selected from an encoded statinecombinatorial library (Carrol et al., 1998). Thus, the combination ofhigh affinity, high selectivity and high biological inhibition activitymakes allophenylnorstatine-based compounds novel and powerful plasmepsinII inhibitors. Specific compounds such as KNI-727 and also providesimportant prototypes for the further development of new antimalarialcompounds by further refinement of their structures.

While the invention been described in terms of its preferred embodimentsskilled in the art will recognize that the invention can be practicedwith modification within the spirit and scope of the appended claims.Accordingly, the present invention should not be limited to theembodiments as described above, but should further include allmodifications and equivalents thereof within the spirit and scope of thedescription provided herein.

REFERENCES

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1. A method of inhibiting a plasmepsin, comprising, exposing saidplasmepsin to an allophenylnorstatine-based compound.
 2. The method ofclaim 1 wherein said plasmepsin is selected from the group consisting ofPlasmepsin I, Plasmepsin II, Plasmepsin IV, and HAP.
 3. The method ofclaim 1 wherein said plasmepsin is Plasmepsin II.
 4. The method of claim1 wherein said allophenylnorstatine-based compound is substituted atpositions R1, R2, R3 and R4 such that R1 is A or A-B, wherein A isselected from the group consisting of a linear or branched aliphatichydrocarbon having 1-7 carbon atoms which may be substituted by at leastone carboxyl group; a 6-membered monocyclic hydrocarbon which may besubstituted with a substituent selected from the group consisting ofalkyl, amino, alkylamino, arylamino, hydroxy, alkloxy and halogen atom;a bicyclic hydrocarbon having 7-10 carbon atoms which may be substitutedby a substituent selected from the group consisting of alkyl, amino,alkylamino, arylamino, hydroxy, alkyloxy and halogen atom; and amonocyclic or bicyclic hydrocarbon wherein more than one carbon atom issubstituted; and B is selected from the group consisting of—CO—NH—CH(Ra)—, —CH₂—CO—NH(Ra)—, —O—CH₂—CO—NH—CH(Ra)—, —OCH₂— and —CH₂O,wherein Ra is a linear or branched aliphatic hydrocarbon having 1-7carbon atoms that may be substituted with a substituent selected fromthe group consisting of alkylthio, hydroxy, aromatic hydrocarbons, andcarbamoyl; R2 is hydrogen or a linear or branched aliphatic hydrocarbonhaving 1-6 carbons; R3 is hydrogen or a linear or branched aliphatichydrocarbon having 1-6 carbons; and R4 is selected from the groupconsisting of a linear or branched aliphatic hydrocarbon having 1-10carbons which can be substituted with a substituent selected from thegroup consisting of aryl, hydroxyl, alkyloxy, amino, alkylamino andhalogen; a monovalent moiety derived from an aromatic mono- or bicyclichydrocarbon having 12 or fewer carbons, and wherein said moiety, can besubstituted by a substituent selected from the group consisting ofalkyl, aryl, hydroxyl, alkyloxy, amino, alkylamino, or halogen; amonovalent moiety derived from a heterocycle in which more than onecarbon atom is substituted with a hetero atom, wherein said moiety canbe substituted by a substituent selected from the group consisting ofalkyl, aryl, hydroxyl, alkyloxy, amino, alkylamino, and halogen.
 5. Themethod of claim 4 wherein A is selected from the group consisting ofHOOC—CRbRb-CRbRb- wherein Rb is hydrogen or methyl; phenyl;3-hydroxy-2-methylphenyl; 2,6-dimethylphenyl; 3-chlorophenyl;3-phenylaminophenyl; 3-dimethylaminophenyl; 1-naphtyl; 2-naphtyl;2-pyridyl; 5-isoquinolyl; 2-quinolyl; 2-benzofuranyl; and 2-chromonyl; Bis selected from the group consisting of —CO—NH—CH(Ra)—,—CH₂—CO—NH(Ra)—, —O—CH₂—CO—NH—CH(Ra)—, —OCH₂— and —CH₂O, wherein Ra ispropyl, isopropyl, isobutyl, sec-butyl, methylthiomethyl,methylthioethyl, ethylthiomethyl, phenylmethyl, carbamoylethyl, or1-hydroxyethyl; R2 is hydrogen or methyl; R3 is hydrogen or methyl; andR4 is selected from the group consisting of benzyl; tert-butyl;2-hydroxybenzyl; 3-hydroxybenzyl; 4-hydroxybenzyl; 2-hydroxyindanylyl;2-hydroxy-1-phenethyl; 1-indanyl; 2-methoxybenzyl; 3-methoxybenzyl;4-methoxybenzyl; 4-methoxyphenethyl; 2-methylbenzyl; 3-methylbenzyl;4-methylbenzyl; naphtyl; and 1-phenethyl.
 6. The method of claim 5wherein said plasmepsin is Plasmepsin II and R1 is selected from thegroup consisting of 2,6-dimethylphenyl-OCH₂—, 3-hydroxy-2 methylphenyland 5-isoquinolyl-O—CH₂—CO—NH—CH(Ra) where Ra is methylthiomethyl; R2 ismethyl; R3 is methyl; and R4 is selected from the group consisting ofbenzyl; tert-butyl; 2-hydroxybenzyl; 3-hydroxybenzyl; (1S,2R)-2-hydroxyindanyl; (1R,2S)-2-hydroxyindanyl;(S)-2-hydroxy-1-phenethyl; (S)-indanyl; 4-methoxyphenethyl;2-methylbenzyl; 3-methylbenzyl; naphthyl; and (R)-1-phenethyl.
 7. Themethod of claim 1 wherein said allophenylnorstatine-based compound is adi-peptide.
 8. The method of claim 7 wherein R1 is selected from thegroup consisting of methylphenol. methylated derivatives of carboxyl,and chlorobenzene.
 9. The method of claim 1 wherein saidallophenylnorstatine-based compound is a tri-peptide.
 10. The method ofclaim 9 wherein R1 is selected from the group consisting ofmethylphenol, methylated derivatives of carboxyl, chlorobenzene, valine,leucine, isoleucine, methionine, phenylalanine, glutamine, andderivatives thereof.
 11. The method of claim 4 wherein R2 and R3 areselected from the group consisting of hydrogen, methyl and ethyl. 12.The method of claim 4 wherein R4 is aminoindanol.
 13. The method ofclaim 4 wherein said allophenylnorstatine-based compound is atri-peptide and: R1 is isoquinolineoxyacetyl at position P3 andmethylthioalanine at position P2, R2 and R3 are methyl, and R4 isselected from the group consisting of (1S,2R)-aminoindanol andtert-butylamine.
 14. The method of claim 1 wherein saidallophenylnorstatine-based compound exhibits a Ki for Plasmepsin II fromP. falciparum in the nanomolar to subnanomolar range.
 15. The method ofclaim 1 wherein said allophenylnorstatine-based compound is selectedfrom the group consisting of KNI-727, KNI-764, KNI-840, KNI-227,KNI-10006, KNI-10026, KNI-10033, KNI-10043 and KNI-10053.
 16. The methodof claim 1 wherein said plasmepsin is Plasmepsin II that originates froma genus of Plasmodium selected from the group consisting of P.falciparum, P. vivax, P. malariae or P. ovale.
 17. A method of killing amalarial parasite comprising, exposing said malarial parasite to anallophenylnorstatine-based compound in a quantity sufficient to inhibitat least one plasmepsin of said malarial parasite and to kill saidmalarial parasite.
 18. The method of claim 17 wherein said malarialparasite is within a red blood cell.
 19. The method of claim 17 whereinsaid malarial parasite is within a host.
 20. The method of claim 17wherein said plasmepsin is selected from the group consisting ofPlasmepsin I, Plasmepsin II, Plasmepsin IV, and HAP.
 21. The method ofclaim 17 wherein said plasmepsin is Plasmepsin II.
 22. The method ofclaim 17 wherein said allophenylnorstatine-based compound is substitutedat positions R1, R2, R3 and R4 such that R1 is A or A-B, wherein A isselected from the group consisting of a linear or branched aliphatichydrocarbon having 1-7 carbon atoms which may be substituted by at leastone carboxyl group; a 6-membered monocyclic hydrocarbon which may besubstituted with a substituent selected from the group consisting ofalkyl, amino, alkylamino, arylamino, hydroxy, alkyloxy and halogen atom;a bicyclic hydrocarbon having 7-10 carbon atoms which may be substitutedby a substituent selected from the group consisting of alkyl, amino,alkylamino, arylamino, hydroxy, alkyloxy and halogen atom; and amonocyclic or bicyclic hydrocarbon wherein more than one carbon atom issubstituted; and B is selected from the group consisting of—CO—NH—CH(Ra)—, —CH₂—CO—NH(Ra)—, —O—CH₂—CO—NH—CH(Ra)—, —OCH₂— and —CH₂O,wherein Ra is a linear or branched aliphatic hydrocarbon having 1-7carbon atoms that may be substituted with a substituent selected fromthe group consisting of alkylthio, hydroxy, aromatic hydrocarbons, andcarbamoyl; R2 is hydrogen or a linear or branched aliphatic hydrocarbonhaving 1-6 carbons; R3 is hydrogen or a linear or branched aliphatichydrocarbon having 1-6 carbons; and R4 is selected from the groupconsisting of a linear or branched aliphatic hydrocarbon having 1-10carbons which can be substituted with a substituent selected from thegroup consisting of aryl, hydroxyl, alkyloxy, amino, alkylamino andhalogen; a monovalent moiety derived from an aromatic mono- or bicyclichydrocarbon having 12 or fewer carbons, and wherein said moiety can besubstituted by a substituent selected from the group consisting ofalkyl, aryl, hydroxyl, alkyloxy, amino, alkylamino, or halogen; amonovalent moiety derived from a heterocycle in which more than onecarbon atom is substituted with a hetero atom, wherein said moiety canbe substituted by a substituent selected from the group consisting ofalkyl, aryl, hydroxyl, alkyloxy, amino, alkylamino, and halogen.
 23. Themethod of claim 17 wherein said allophenylnorstatine-based compound isselected from the group consisting of KNI-727, KNI-764, KNI-840,KNI-227, KNI-10006, KNI-10026, KNI-10033, KNI-10043 and KNI-10053. 24.The method of claim 17 wherein said malarial parasite is selected fromthe group consisting of P. falciparum, P. viva, P. malariae or P. ovale.25. A method of treating malaria in a patient in need thereof,comprising, administering to said patient a quantity of anallophenylnorstatine-based compound sufficient to alleviate symptoms ofsaid malaria.
 26. The method of claim 25 wherein saidallophenylnorstatine-based compound is substituted at positions R1, R2,R3 and R4 such that R1 is A or A-B, wherein A is selected from the groupconsisting of a linear or branched aliphatic hydrocarbon having 1-7carbon atoms which may be substituted by at least one carboxyl group; a6-membered monocyclic hydrocarbon which may be substituted with asubstituent selected from the group consisting of alkyl, amino,alkylamino, arylamino, hydroxy, alkyloxy and halogen atom; a bicyclichydrocarbon having 7-10 carbon atoms which may be substituted by asubstituent selected from the group consisting of alkyl, amino,alkylamino, arylamino, hydroxy, alkyloxy and halogen atom; and amonocyclic or bicyclic hydrocarbon wherein more than one carbon atom issubstituted; and B is selected from the group consisting of—CO—NH—CH(Ra)—, —CH₂—CO—NH(Ra)—, —O—CH₂—CO—NH—CH(Ra)—, —OCH₂— and —CH₂O,wherein Ra is a linear or branched aliphatic hydrocarbon having 1-7carbon atoms that may be substituted with a substituent selected fromthe group consisting of alkylthio, hydroxy, aromatic hydrocarbons, andcarbamoyl; R2 is hydrogen or a linear or branched aliphatic hydrocarbonhaving 1-6 carbons; R3 is hydrogen or a linear or branched aliphatichydrocarbon having 1-6 carbons; and R4 is selected from the groupconsisting of a linear or branched aliphatic hydrocarbon having 1-10carbons which can be substituted with a substituent selected from thegroup consisting of aryl, hydroxyl, alkyloxy, amino, alkylamino andhalogen; a monovalent moiety derived from an aromatic mono- or bicyclichydrocarbon having 12 or fewer carbons, and wherein said moiety can besubstituted by a substituent selected from the group consisting ofalkyl, aryl, hydroxyl, alkyloxy, amino, alkylamino, or halogen; amonovalent moiety derived from a heterocycle in which more than onecarbon atom is substituted with a hetero atom, wherein said moiety canbe substituted by a substituent selected from the group consisting ofalkyl, aryl, hydroxyl, alkyloxy, amino, alkylamino, and halogen.
 27. Themethod of claim 25 wherein said allophenylnorstatine-based compound isselected from the group consisting of KNI-727, KNI-764, KNI-840,KNI-227, KNI-10006, KNI-10026, KNI-10033, KNI-10043 and KNI-10053. 28.The method of claim 25 wherein said allophenylnorstatine-based compoundhas high selectivity for plasmepsin rather than Cathepsin D.
 29. Amethod of inhibiting the enzyme Cathepsin D, comprising, exposing saidenzyme to an allophenylnorstatine-based compound. cm
 30. The method ofclaim 29 wherein said allophenylnorstatine-based compound is substitutedat positions R1, R2, R3 and R4 wherein R1 is 2,6-dimethylphenyl-OCH₂— or5-isoquinolyl-O—CH₂—CO—NH—CH(Ra)— wherein Ra is methylthiomethyl; R2 ismethyl or hydrogen; R3 is methyl or hydrogen; and R4 is selected fromthe group consisting of (1S, 2R)-2-hydroxyindanyl;(S)-2-hydroxy-1-phenethyl; (S)-indanyl; and (R)-1-phenethyl.
 31. Themethod of claim 29 wherein said allophenylnorstatine-based compound issubstituted at positions R1, R2, R3 and R4 wherein when saidallophenylnorstatine-based compound is a dipeptide R1 is carboxyl or aderivative thereof at position P2 and when saidallophenylnorstatine-based compound is a tripeptide R1 is chromen-4-oneat position P3 or valine at position P3; R2 and R3 are hydrogen; and R4is selected from the group consisting of indanol and tert-butylamine.32. The method of claim 29 wherein said allophenylnorstatine-basedcompound is selected from the group consisting of KNI-391, KNI-10033,KNI-10006 and KNI-840.
 33. KNI-10006
 34. KNI-10007
 35. KNI-10026 36.KNI-10031
 37. KNI-10061.
 38. KNI-10062.